Sample quality requirements:

Genomic DNA should be intact. If your DNA sample is degraded please contact us to coordinate suitable sample prep.
RNA integrity must be confirmed using the Agilent Bioanalyzer/ TapeStation/ similar instrument, or by running the sample on an agarose gel.
Sample purity:
OD260/280 = 1.8-2.2
OD260/230 ≥ 2.0

Library Preparation

Library preparation is the process by which an initial sample, typically genomic DNA or total RNA, is processed to become a library ready for sequencing.

In general, DNA is sheared randomly, creating blunt- end fragments. Blunt ends are then adenylated in preparation for adaptor ligation. Adaptors contain sample-specific indexes to individually tag each sample. Size-specific magnetic beads are used for fragment size selection. Enrichment of adaptor-bound inserts is then achieved by PCR amplification, thereby enabling sample quantification for loading onto the sequencer. Samples prepared from RNA are usually subjected to poly-A selection in order to select for mRNA specifically to be prepared for sequencing. RNA is fragmented, reverse transcribed to cDNA, and then undergoes a similar process to that of DNA sample preparations.

The adaptors that were ligated to fragments during sample preparation hybridize to the flowcell on which they are sequenced. These adaptors contain a unique 6-8bp sequence, known as an “index” or “barcode”, essentially “tagging” each individual sample and enabling for multiple samples to be sequenced together in a pool. Because index sequences are unique, individual samples are then able to be identified according to their assigned index during bioinformatic analyses.